Systems and methods for generating immune responses in subjects using microchannel delivery devices

ABSTRACT

The present invention provides a method for generating an immune response in a subject, comprising administering to the subject&#39;s skin an immunizing composition from a SARS-CoV-2 pathogen, wherein the composition is administered with a microneedle delivery device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. No.62/992,810, filed on Mar. 20, 2020, and U.S. Provisional Appl. No.62/986,539, filed Mar. 6, 2020, the contents of which are herebyincorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablesequence listing submitted concurrently herewith and identified asfollows: One 137,944 Byte ASCII (Text) file named“sequence_listing.txt,” created on Aug. 17, 2020.

FIELD OF THE INVENTION

The field of the invention relates generally to the field of medicine,medical devices, immunology, and infectious disease, specificallymethods and devices useful for generating immune responses in subjects.

SUMMARY OF THE INVENTION

It is to be understood that both the foregoing general description ofthe embodiments and the following detailed description are exemplary,and thus do not restrict the scope of the embodiments.

In one aspect, the invention provides a method for generating an immuneresponse in a subject, comprising administering to the subject's skin animmunizing composition, wherein the composition is administered with amicroneedle delivery device.

In some embodiments, the immunizing composition comprises a heat killedor attenuated pathogen.

In some embodiments, the pathogen is Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2). In some embodiments, the polypeptidecomprises any one of or a combination of SEQ ID NOS:1-12, or anantigenic fragment or derivative thereof. In some embodiments, theantigen comprises a fragment of SEQ ID NO:4. In some embodiments, thepolypeptide comprises amino acids 330 to 521 of SEQ ID NO:4.

In some embodiments, the administering comprises a repeated motion ofpenetrating the microneedle delivery device into the subject's skin.

In some embodiments, the administering comprises a repeated motion ofpenetrating the microneedle delivery device into the subject's skin indifferent areas of the subject's body. In some embodiments, a protectiveimmune response is achieved after one administration. In someembodiments, one or more booster administrations are administered toachieve a protective immune response.

In some embodiments, the subject's skin in the head, limbs and/or torsoregions are repeatedly penetrated by the microneedle delivery device.

In some embodiments, the subject's skin is penetrated in regions thatare in proximity to one or more lymph nodes.

In some embodiments, the subject administers the immunizing compositionto his or her own skin.

In some embodiments, the microneedle delivery device comprises

i) a plurality of microneedles, wherein the microneedles are hollow ornon-hollow, wherein one or multiple grooves are inset along an outerwall of the microneedles; and

ii) a reservoir that holds the composition to be delivered, wherein thereservoir is attached to or contains a means to encourage flow of thecomposition contained in the reservoir into the skin;

wherein the composition is delivered into the skin by passing throughthe one or multiple grooves along the outer wall of the microneedle.

In some embodiments, the microneedles are non-hollow.

In some embodiments, the means to encourage flow of the compositioncontained in the reservoir into the skin is selected from the groupconsisting of a plunger, pump and suction mechanism.

In some embodiments, the means to encourage flow of the compositioncontained in the reservoir into the skin is a mechanical spring-loadedpump system.

In some embodiments, the microneedles have a single groove inset alongthe outer wall of the microneedle, wherein the single groove has a screwthread shape going clockwise or counterclockwise around the microneedle.

In some embodiments, the microneedles are from 0.1 mm to about 2.5 mm inlength and from 0.01 mm to about 0.05 mm in diameter.

In some embodiments, the microneedles are made from a substancecomprising gold.

In some embodiments, the plurality of microneedles comprises an array ofmicroneedles in the shape of a circle.

In some embodiments, the microneedles are made of 24-carat gold platedstainless steel and comprise an array of 20 microneedles.

In some embodiments, the devices and methods enable non-medicalprofessionals to administer therapeutics, including the capability forself-administration during times of epidemic and pandemic crises (suchas SARS, MERS, SARS-CoV-2 (COVID-19), etc.), when there is no or limitedaccess to healthcare providers and healthcare systems are strained orlimited.

In some embodiments, the apparatus is user-friendly and self-explanatory(or with minimum learning curve) for ease of use by users andindividuals without scientific or medical backgrounds.

In one aspect, the invention provides a single-use dermal drug deliveryapparatus that aids in a method of self-administration or administrationby non-medical professionals of therapeutic agents, such as biologics,drugs, vaccines, etc. In accordance with the invention, the devicecomprises a microchannel or microneedle delivery apparatus.

Unlike syringe needles, in some embodiments the microneedle systems ofthe invention are painless, user-friendly and requires a low learningcurve. In some embodiments, the devices can be used by individualsduring pandemic/endemic situations or at times when access to healthcareproviders and institutions is restricted or limited. It can also beadministered by registered physicians, nurses and affiliated healthcareproviders.

In some embodiments, the device comprises a housing comprising a lockand break mechanism which enables it to be a single-use apparatus. Forexample, once the drug administration is completed, the apparatusbecomes non-functional. In some embodiments, if the apparatus is forcedto be reused, it can break.

In many instances, it will be desirable to have multiple administrationsof a therapeutic compositions, such as a vaccine or immunizingcomposition. In some embodiments, in the case of vaccinations, thesubjects are usually not administered more than six vaccinations. Incases of multiple administrations (e.g., booster vaccinations), multiplesingle-use housings and multiple cartridges can be provided. In someembodiments, after each administration, the housing can be replaced byanother housing, and the cartridge can be replaced by a new cartridge.

In some embodiments, the devices can be delivered directly to consumers,as single use devices, multiple use devices, or batches of devices inorder to provide devices for members of a community or family. Forexample, the devices can be delivered to individual houses, cities andpick-up locations. In some embodiments, the devices can bemass-distributed and dispatched through aerial vehicles such ashelicopters, drones, and other unmanned aerial vehicles.

In some embodiments, the devices can be marked with a modular smartlabel data transmission system for applied end-use optimization. Thishelps with tracking of individual devices and helps collect data andfeedback to optimize user experience, operations, and supply logistics.

In another aspect, the invention provides a method for generating animmune response in a subject, comprising administering to the subject'sskin an immunizing composition, wherein the composition is administeredwith a microneedle delivery device.

In some embodiments, the immunizing composition comprises an inactivatedor attenuated pathogen.

In some embodiments, the immunizing composition comprises animmunologically effective amount of one or more polypeptides from apathogen or antigenic fragments or variants thereof.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a view of a handheld microneedle injection apparatus. Thesyringe ejection volume is automatically controlled and dispenses intoan interchangeable head containing one or several needles. The diagramshows the connection of corrugated connector and microneedle head. Therubber-based connector is such that its flexibility will allowconnections with small openings (1) and large ones (2) to fit and sealthe microneedle head. The corrugated connector, also made of rubber (3),will further allow larger embodiments to connect to this system with thespring plate microneedle head (4).

FIG. 2 is an image of microneedles on a microneedle head.

FIG. 3 is a schematic representation of a device in a syringeconfiguration. Alternative configurations include vial- andcapsule-loaded configurations. The device holds a syringe (2) forautomatic injection via a plurality of microneedles in the microneedlehead. Ejection volume is controlled by an information processor (9).Other elements are noted: the motor or actuator (4) to control thepiston (3), exchangeable and controllable needle head (1) and cam systemand dial to adjust needle injection depth (5), and needle head ejector(10). Information is shown to the user in a display panel that mayinclude a manual or touchscreen control panel (12) and data is stored ina storage unit (11) that may be removable. The needle head (1) may becontrolled by an actuator (13).

FIG. 4 provide three additional views of a microneedle device.Microneedle components: (A) microneedles, (B) housing of the needles and(C) a reservoir.

FIG. 5 provides a depiction of the utility feature conferred by thecircular or flat O-Rings. Said features enable enhanced liquid handlingcapabilities as evidenced by an airtight mechanism which facilitates theefficient and uniform delivery of treatment solutions to the skin. Saidfeatures are positioned at the interface of the cap and the reservoirchannel so as to effectively prevent the leakage of treatment solutiondosages. The RFID chip+O-ring depiction has been expanded. The cap/cover(1) will interface with the vial or container (5) containing a certaincompound (6). The connection of both the cap/cover and the container maybe sealed with a threaded opening (2). While pressure is appliedvertically through the twisting motion of the thread, the rubber O-ring(3) seals the two interfaces (1) and (5) together. A ratchet mechanism(4) at the end will lock the cap in place. Embedded inside the rubberO-ring is a RFID chip (7) which material is shock, pH, temperature, andozone resistant. The RFID chip will be stable enough under differentenvironments to be able to effectively transmit data for applicationssuch as data security, quality assurance/control, and logistics (8). TheRFID chip will enable tracking usage of the microchannel device and willbe connected with a cloud data. It will transmit information on theusage of device, attempt to reuse, and status of device functionality.This information will be shared with the subject to keep track of dateof administration and notifying date of booster administration.

FIG. 6 illustrates anti-unlock safety features of an O ring in amicroneedle device.

FIG. 7 illustrates an exemplary microneedle drug delivery device.

FIG. 8 illustrates an exemplary microneedle drug delivery device.

FIG. 9 illustrates an exemplary microneedle drug delivery device.

FIG. 10 illustrates a cap element of an exemplary microneedle drugdelivery device.

FIG. 11 illustrates a housing element of an exemplary microneedle drugdelivery device.

FIG. 12. illustrates a cartridge element of an exemplary microneedledrug delivery device.

FIG. 13. Mass distribution of devices.

FIG. 14 illustrates a lock-break mechanism of the microneedle drugdelivery device wherein as soon as the subject pushes the pusher, itbreaks the lock plate 1 and renders the device non-reusable. The lockplate II is a secondary break mechanism system that also renders thedevice non-reusable. Because of the lock-break mechanisms, the subjectcan only administer with the device once. The microneedle heads andreservoir assembly in the device are modular and detachable.

FIG. 15 illustrates a lock-break mechanism of the microneedle drugdelivery device wherein as soon as the subject pushes the plunger, itbreaks the lock plate 1 and renders the device non-reusable. The lockplate II is a secondary break mechanism system that also renders thedevice non-reusable. Because of the lock-break mechanisms, the subjectcan only administer with the device once. The microneedle heads andreservoir assembly in the device are modular and detachable.

FIG. 16 illustrates a multi chamber microneedle drug delivery devicedesign that features a pusher that is activated by the subject. Thepusher pierces the layer separating chamber I and chamber II therebyallowing the flow of bioactive composition from chamber I to chamber II.After this, the bioactive compositions are mixed by a gravity-drivenmotion by shaking the device. After this, the bioactive compositiontransfers to the reservoir and can be administered on a subject. Themicrochannel head facilitates movement from the reservoir to thesubject's skin.

FIG. 17 illustrates a multi chamber microneedle drug delivery devicedesign that features a pusher that is activated by the subject. Thepusher pierces the layer separating chamber I and chamber II therebyallowing the flow of bioactive composition from chamber I to chamber II.After this, the bioactive compositions are mixed by a gravity-drivenmotion by shaking the device. After this, the bioactive compositiontransfers to the reservoir and can be administered on a subject. Themicrochannel head facilitates movement from the reservoir to thesubject's skin.

FIG. 18 illustrates a modular multi chamber microneedle drug deliverydevice design. This allows the chambers and the reservoir with themicroneedle head to be detachable. The chambers can be replaced orsubstituted. It features a pusher that is activated by the subject. Thepusher pierces the layer separating Chamber I and Chamber II therebyallowing the flow of bioactive composition from chamber I to chamber II.After this, the bioactive compositions are mixed by a gravity-drivenmotion by shaking the device. After this, the bioactive compositiontransfers to the reservoir and can be administered on a subject. Themicrochannel head facilitates movement from the reservoir to thesubject's skin.

FIG. 19 illustrates a multi chamber microneedle drug delivery devicedesign that features a pusher that is activated by the subject. Thepusher pierces the layer separating Chamber I and Chamber II therebyallowing the flow of bioactive composition from chamber I to chamber II.After this, the bioactive compositions are mixed by a gravity-drivenmotion by shaking the device. After this, the bioactive compositiontransfers to the reservoir and can be administered on a subject. Themicrochannel head facilitates movement from the reservoir to thesubject's skin. It also features a blender that can be activated by thesubject through an external button/switch. This blender helps in mixingthe bioactive composition.

FIG. 20 illustrates a multi chamber microneedle drug delivery devicedesign that features multiple pushers that are activated individually ortogether by the subject. Each pusher pierces the layer separating thetwo chambers thereby allowing the flow of bioactive composition from onechamber to another. After this, the bioactive compositions are mixed bya gravity-driven motion by shaking the device. After this, the bioactivecomposition transfers to the reservoir and can be administered on asubject. The microchannel head facilitates movement from the reservoirto the subject's skin. Each of these chambers can contain differentcompositions.

FIG. 21 illustrates a modular multi chamber microneedle drug deliverydevice design that features multiple chambers that can be attached toeach other. Each chamber features a pusher that pierces the layerseparating the two chambers thereby allowing the flow of bioactivecomposition from one chamber to another. After this, the bioactivecompositions are mixed by a gravity-driven motion by shaking the device.After this, the bioactive composition transfers to the reservoir and canbe administered on a subject. The microchannel head facilitates movementfrom the reservoir to the subject's skin. Each of these chambers cancontain different compositions.

FIG. 22 illustrates a modular multi chamber microneedle drug deliverydevice design that features two chambers that can be attached to eachother wherein one chamber contains the pusher that pierces the otherchamber. The pusher pierces the outer layer of the attached chamberthereby allowing flow of bioactive composition from one chamber toanother. After this, the bioactive compositions are mixed by agravity-driven motion by shaking the device. After this the bioactivecomposition transfers to the reservoir and can be administered on asubject. The microchannel head facilitates movement from the reservoirto the subject's skin. Each of these chambers can contain differentcompositions.

FIG. 23 illustrates the process of self-administration device andprocess.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention which, together with the drawings and thefollowing examples, serve to explain the principles of the invention.These embodiments are described in sufficient detail to enable thoseskilled in the art to practice the invention, and it is understood thatother embodiments may be utilized, and that structural, biological, andchemical changes may be made without departing from the spirit and scopeof the present invention. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Sambrook et al. MolecularCloning: A Laboratory Manual, 2^(nd) edition (1989); Current Protocolsin Molecular Biology (F. M. Ausubel et al. eds. (1987)); the seriesMethods in Enzymology (Academic Press, Inc.); PCR: A Practical Approach(M. MacPherson et al. IRL Press at Oxford University Press (1991)); PCR2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Tayloreds. (1995)); Antibodies, A Laboratory Manual (Harlow and Lane eds.(1988)); Using Antibodies, A Laboratory Manual (Harlow and Lane eds.(1999)); and Animal Cell Culture (R. I. Freshney ed. (1987)).

Definitions of common terms in molecular biology may be found, forexample, in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341).

For the purpose of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). The use of “or” means“and/or” unless stated otherwise. As used in the specification andclaims, the singular form “a,” “an” and “the” include plural referencesunless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof. The useof “comprise,” “comprises,” “comprising,” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.Furthermore, where the description of one or more embodiments uses theterm “comprising,” those skilled in the art would understand that, insome specific instances, the embodiment or embodiments can bealternatively described using the language “consisting essentially of”and/or “consisting of.”

“Apparatus” and “device” are used interchangeably herein.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used.

In one embodiment, the invention provides a method for generating animmune response in a subject, comprising administering to the subject'sskin an immunizing composition, wherein the composition is administeredwith a microneedle delivery device.

The term “subject” as used herein is not limiting and is usedinterchangeably with patient. In some embodiments, the term subjectrefers to animals, such as mammals and the like. For example, mammalscontemplated include humans, primates, dogs, cats, sheep, cattle, goats,pigs, horses, chickens, mice, rats, rabbits, guinea pigs, and the like.

In some embodiments, the subject is a human and administers theimmunizing composition to his or her own skin.

In some embodiments, the immunizing composition is derived from apathogen. In some embodiments, the pathogen is a bacterial or viralpathogen. In some embodiments, the pathogen is selected from the groupconsisting of Streptococcus pneumonia, Neisseria meningitidis,Haemophilus influenza, Klebsiella spp., Pseudomonas spp., Salmonellaspp., Shigella spp., and Group B streptococci, Bacillus anthracisadenoviruses; Bordetella pertussus; Botulism; bovine rhinotracheitis;Brucella spp.; Branhamella catarrhalis; canine hepatitis; caninedistemper; Chlamydiae; Cholera; coccidiomycosis; cowpox; tularemia;filoviruses; arenaviruses; bunyaviruses; cytomegalovirus;cytomegalovirus; Dengue fever; dengue toxoplasmosis; Diphtheria;encephalitis; Enterotoxigenic Escherichia coli; Epstein Barr virus;equine encephalitis; equine infectious anemia; equine influenza; equinepneumonia; equine rhinovirus; feline leukemia; flavivirus; Burkholderiamallei; Globulin; Haemophilus influenza type b; Haemophilus influenzae;Haemophilus pertussis; Helicobacter pylori; Haemophilus spp.; hepatitis;hepatitis A; hepatitis B; Hepatitis C; herpes viruses; HIV; HIV-1viruses; HIV-2 viruses; HTLV; Influenza; Japanese encephalitis;Klebsiellae spp. Legionella pneumophila; Leishmania; leprosy; lymedisease; malaria immunogen; measles; meningitis; meningococcal;Meningococcal Polysaccharide Group A, Meningococcal Polysaccharide GroupC; mumps; Mumps Virus; mycobacteria; Mycobacterium tuberculosis;Neisseria spp; Neisseria gonorrhoeae; ovine blue tongue; ovineencephalitis; papilloma; SARS and associated coronaviruses; Severe AcuteRespiratory Syndrome Coronavirus 2 (SARS-CoV-2) (COVID-19);parainfluenza; paramyxovirus; paramyxoviruses; Pertussis; Plague;Coxiella burnetti; Pneumococcus spp.; Pneumocystis carinii; Pneumonia;Poliovirus; Proteus species; Pseudomonas aeruginosa; rabies; respiratorysyncytial virus; rotavirus; Rubella; Salmonellae; schistosomiasis;Shigellae; simian immunodeficiency virus; Smallpox; Staphylococcusaureus; Staphylococcus spp.; Streptococcus pyogenes; Streptococcus spp.;swine influenza; tetanus; Treponema pallidum; Typhoid; Vaccinia;varicella-zoster virus; and Vibrio cholera and combinations thereof.

In some embodiments, the immunizing composition comprises a heat killedor attenuated pathogen.

In some embodiments, the immunizing composition comprises animmunologically-effective amount of one or more polypeptides from apathogen or antigenic fragments or variants thereof.

In some embodiments, the immunizing composition comprises animmunologically-effective amount of a nucleic acid encoding apolypeptide or an antigenic fragment or variant thereof from a pathogen.In some embodiments, the nucleic acid is a DNA. In some embodiments, thenucleic acid is an mRNA.

In some embodiments, the pathogen is Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2).

In some embodiments, the immunizing composition comprises a heat killedor attenuated SARS-CoV-2, a polypeptide or an antigenic fragment orvariant thereof from SARS-CoV-2, or a nucleic acid encoding the same. Insome embodiments, the antigen is the spike protein or an antigenicfragment or variant thereof from SARS-CoV-2. In some embodiments, thespike protein has the sequence found in GenBank accession no.:QIC53213.1. In some embodiments, the spike protein has the sequencefound in any of SEQ ID NOS:1-12. In some embodiments, the antigen is thefull-length SARS-CoV-2 spike protein.

In some embodiments, the antigen comprises all or part of thereceptor-binding domain (RBD) of the SARS-CoV-2 spike protein or afragment or variant thereof, such as a deglycosylated version. In someembodiments, the RBD of the SARS-CoV-2 spike protein comprises aminoacids 330 to 521 of SEQ ID NO:4.

In some embodiments, the antigen is encoded by SEQ ID NO:13, or afragment thereof.

In some embodiments, the immunizing composition comprises a nucleic acidencoding an antigenic polypeptide. In some embodiments, the nucleic acidcomprises SEQ ID NO:13 or a fragment thereof. In some embodiments, theantigenic polypeptide comprises a SARS-CoV-2 spike protein or a fragmentthereof. In some embodiments, the antigenic polypeptide comprises aminoacids 330 to 521 of SEQ ID NO:4. In some embodiments, the nucleic acidis a mRNA. In some embodiments, the mRNA is formulated in ananoparticle, such as a solid lipid nanoparticle.

In some embodiments, the nucleic acid is delivered by a viral vector. Insome embodiments, the viral vector comprises a nucleic acid sequenceencoding the SARS-CoV-2 spike protein or an antigenic fragment orderivative thereof. In some embodiments, the spike protein or anantigenic fragment or derivative thereof is fused to an epitope tag. Theepitope tag is not limiting, and in some embodiments is selected fromthe group consisting of Myc, FLAG, hemagglutinin (HA) and/orcombinations thereof. In some embodiments, the spike protein or anantigenic fragment or derivative thereof encodes a protein that is atleast 90% identical to SEQ ID NO:4.

The viral vector is not limiting. In some embodiments, the viral vectorwill typically comprise a highly attenuated, non-replicative virus.Viral vectors include, but are not limited to, DNA viral vectors such asthose based on adenoviruses, herpes simplex virus, avian viruses, suchas Newcastle disease virus, poxviruses such as vaccinia virus, andparvoviruses, including adeno-associated virus; and RNA viral vectors,including, but not limited to, the retroviral vectors. Vaccinia vectorsand methods useful in immunization protocols are described in U.S. Pat.No. 4,722,848. Retroviral vectors include murine leukemia virus, andlentiviruses such as human immunodeficiency virus. Naldini et al. (1996)Science 272:263-267. Replication-defective retroviral vectors harboringa nucleotide sequence of interest as part of the retroviral genome canbe used. Such vectors have been described in detail. (Miller et al.(1990) Mol. Cell. Biol. 10:4239; Kolberg, R. (1992) J. NIH Res. 4:43;Cornetta et al. (1991) Hum. Gene Therapy 2:215).

Adenovirus and adeno-associated virus vectors useful in the inventionmay be produced according to methods already taught in the art. (See,e.g., Karlsson et al. (1986) EMBO 5:2377; Carter (1992) Current Opinionin Biotechnology 3:533-539; Muzcyzka (1992) Current Top. Microbiol.Immunol. 158:97-129; Gene Targeting: A Practical Approach (1992) ed. A.L. Joyner, Oxford University Press, NY). Several different approachesare feasible.

Alpha virus vectors, such as Venezuelan Equine Encephalitis (VEE) virus,Semliki Forest virus (SFV) and Sindbis virus vectors, can be used forefficient gene delivery. Replication-deficient vectors are available.Such vectors can be administered through any of a variety of means knownin the art, such as, for example, intranasally or intratumorally. SeeLundstrom, Curr. Gene Ther. 2001 1:19-29.

Additional literature describing viral vectors which could be used inthe methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.)Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F.et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: GeneTransfer and Expression Protocols, Murray, E. (ed.), Humana Press,Clifton, N.J. (1991); Miller, et al. (1995) FASEB Journal 9:190-199,Schreier (1994) Pharmaceutica Acta Helvetiae 68:145-159; Schneider andFrench (1993) Circulation 88:1937-1942; Curiel, et al. (1992) Human GeneTherapy 3:147-154; WO 95/00655; WO 95/16772; WO 95/23867; WO 94/26914;WO 95/02697 (Jan. 26, 1995); and WO 95/25071.

In some embodiments, the viral vector is a retrovirus/lentivirus,adenovirus, adeno-associated virus, alpha virus, vaccinia virus or aherpes simplex virus. In some embodiments, the viral vector is alentiviral vector comprising the nucleotide sequence of SEQ ID NO:13 ora fragment thereof.

In some embodiments, the method further comprises assaying a sample fromthe subject after administering the immunizing composition. In someembodiments, the assaying comprises detecting the presence of a pathogenusing nucleic acid amplification tests which will determine if thesubject is actively infected by a pathogen. In some embodiments, theassaying comprises detecting the presence of an immune response in thesubject against the immunizing composition. In some embodiments, thedetection is performed by serology tests. In some embodiments, theserology tests are performed with the help of microchannels by isolatinga blood sample and running micro-ELISA tests. In some embodiments,microfluidic systems are employed to run serology tests. In someembodiments, the subjects can perform the diagnostic testing bythemselves.

An antigenic fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof one of the polypeptides. The antigenic fragment can be“free-standing,” or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region.

In some embodiments, the antigenic fragments include, for example,truncation polypeptides having the amino acid sequence of thepolypeptides, except for deletion of a continuous series of residuesthat includes the amino terminus, or a continuous series of residuesthat includes the carboxyl terminus or deletion of two continuous seriesof residues, one including the amino terminus and one including thecarboxyl terminus. In some embodiments, fragments are characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, and high antigenic index regions.

The fragment can be of any size. An antigenic fragment is capable ofinducing an immune response in a subject or be recognized by a specificantibody. In some embodiments, the fragment corresponds to anamino-terminal truncation mutant. In some embodiments, the number ofamino terminal amino acids missing from the fragment ranges from 1-100amino acids. In some embodiments, it ranges from 1-75 amino acids, 1-50amino acids, 1-40 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20amino acids, 1-15 amino acids, 1-10 amino acids and 1-5 amino acids.

In some embodiments, the fragment corresponds to carboxyl-terminaltruncation mutant. In some embodiments, the number of carboxyl terminalamino acids missing from the fragment ranges from 1-100 amino acids. Insome embodiments, it ranges from 1-75 amino acids, 1-50 amino acids,1-40 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids,1-15 amino acids, 1-10 amino acids and 1-5 amino acids.

In some embodiments, the fragment corresponds to an internal fragmentthat lacks both the amino and carboxyl terminal amino acids. In someembodiments, the fragment is 7-200 amino acid residues in length. Insome embodiments, the fragment is 10-100 amino acid residues, 15-85amino acid residues, 25-65 amino acid residues or 30-50 amino acidresidues in length. In some embodiments, the fragment is 7 amino acids,10 amino acids, 12 amino acids, 15 amino acids, 20 amino acids, 25 aminoacids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids,50 amino acids 55 amino acids, 60 amino acids, 80 amino acids or 100amino acids in length.

In some embodiments, the fragment is at least 50 amino acids, 100 aminoacids, 150 amino acids, 200 amino acids or at least 250 amino acids inlength. Of course, larger antigenic fragments are also useful accordingto the present invention, as are fragments corresponding to most, if notall, of the amino acid sequence of the polypeptides described herein.

In some embodiments, the polypeptides have an amino acid sequence atleast 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the polypeptides described herein or antigenic fragmentsthereof. In some embodiments, the variants are those that vary from thereference by conservative amino acid substitutions, i.e., those thatsubstitute a residue with another of like characteristics. Typicalsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg, or aromatic residues Phe and Tyr. In someembodiments, the polypeptides are variants in which several, 5 to 10, 1to 5, or 1 to 2 amino acids are substituted, deleted, or added in anycombination.

In some embodiments, the polypeptides are encoded by polynucleotidesthat are optimized for high level expression in E. coli using codonsthat are preferred in E. coli. As used herein, a codon that is“optimized for high level expression in Salmonella” refers to a codonthat is relatively more abundant in E. coli in comparison with all othercodons corresponding to the same amino acid. In some embodiments, atleast 10% of the codons are optimized for high level expression in E.coli. In some embodiments, at least 25%, at least 50%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99% ofthe codons are optimized for high level expression in E. coli.

In some embodiments, the polypeptide or antigenic fragment thereofcomprises a cleavable protein sequence and/or affinity tag to aid inpurification. In some embodiments, the affinity tag comprises at least 6histidine residues. In some embodiments, the polypeptide or antigenicfragment thereof comprises a secretion signal to facilitate secretion ofthe protein through plasma membrane. In some embodiments, the secretionsignal is a lysozyme secretion signal.

In some embodiments, the compositions are administered as pharmaceuticalcompositions and induce an immune response to the antigen in a cell,tissue or animal (e.g., a human). As used herein, an “antigeniccomposition” (which alternatively may be referred to as an “immunizingcomposition”) may comprise an antigen (e.g., a protein, peptide, orpolypeptide). In some embodiments, the antigenic composition comprises anucleic acid encoding a polypeptide antigen.

In some embodiments, the immunogenic composition or vaccine comprises atleast one adjuvant. In other embodiments, the antigenic composition isin a mixture that comprises an additional immunostimulatory agent ornucleic acids encoding such an agent. Immunostimulatory agents includebut are not limited to an additional antigen, an immunomodulator, anantigen presenting cell or an adjuvant. In other embodiments, one ormore of the additional agent(s) is covalently bonded to the antigen oran immunostimulatory agent, in any combination. In certain embodiments,the antigenic composition is conjugated to or comprises an HLA anchormotif amino acids.

In certain embodiments, an antigenic composition can be used as aneffective vaccine in inducing an anti-SARS-CoV-2 humoral and/orcell-mediated immune response in an animal, including a human. Thepresent invention contemplates one or more antigenic compositions orvaccines for use in both active and passive immunization embodiments.

A vaccine or immunizing composition of the present invention may vary inits composition of proteinaceous components. It will be understood thatvarious compositions described herein may further comprise additionalcomponents. For example, one or more vaccine or immunogenic compositioncomponents may be comprised in a lipid or liposome. In a non-limitingexample, a vaccine or immunogenic composition may comprise one or moreadjuvants. In another non-limiting example, a vaccine or immunogeniccomposition may comprise a saponin and a lipid. A vaccine or immunizingcomposition of the present disclosure, and its various components, maybe prepared by any method disclosed herein or as would be known to oneof ordinary skill in the art, in light of the present disclosure.

It is understood that an immunizing composition may be made by a methodthat is well known in the art, including but not limited to chemicalsynthesis by solid phase synthesis and purification away from the otherproducts of the chemical reactions by HPLC, or production by theexpression of a nucleic acid sequence (e.g., a DNA sequence) encoding apeptide or polypeptide comprising an antigen of the present invention inan in vitro translation system or in a living cell including, forexample, in a yeast cell, bacterial, mammalian cells orbaculovirus/insect cells. The antigenic composition may be isolated andextensively purified to remove one or more undesired small molecularweight molecules and/or lyophilized for more ready formulation into adesired vehicle. It is further understood that amino acid additions,deletions, mutations, chemical modification and such like that are madein an antigenic composition component, such as a vaccine, willpreferably not substantially interfere with the antibody recognition ofthe epitopic sequence.

In some embodiments, a peptide or polypeptide corresponding to one ormore antigenic determinants of the receptor binding domain of theSARS-CoV-2 spike protein may generally be 10-20 amino acid residues inlength, and may contain more than one peptide determinants or up toabout 30-50 residues or so. In some embodiments, the polypeptide isbetween 10 and about 150 residues or more in length. A peptide sequencemay be made by methods known to those of ordinary skill in the art, suchas, for example, peptide synthesis using automated peptide synthesismachines, such as those available from Applied Biosystems (Foster City,Calif.).

In some embodiments, longer peptides or polypeptides also may beprepared, e.g., by recombinant means. In certain embodiments, a nucleicacid encoding an antigenic composition and/or a component describedherein may be used, for example, to produce an antigenic composition invitro or in vivo for the various compositions and methods of the presentinvention. For example, in certain embodiments, a nucleic acid encodingan antigen is comprised in, for example, a vector in a recombinant cell.The nucleic acid may be expressed to produce a peptide or polypeptidecomprising an antigenic sequence. The peptide or polypeptide may besecreted from the cell or comprised as part of or within the cell.

In some embodiments, the antigen may be expressed using a vector such asa viral vector. For example, in certain embodiments, the coding sequencecould be inserted in a viral vector, including but not limited to anadenovirus, adeno-associated virus, measles virus, poxvirus, herpescomplex, retrovirus, lentivirus, alphavirus, flavivirus, rabdovirus,Newcastle disease virus and picronavirus.

As modifications and changes may be made in the structure of anantigenic composition of the present disclosure, and still obtainmolecules having like or otherwise desirable characteristics, suchimmunologically functional equivalents are also encompassed within thepresent invention.

For example, certain amino acids may be substituted for other aminoacids in a peptide, polypeptide or protein structure without appreciableloss of interactive binding capacity with structures such as, forexample, antigen-binding regions of antibodies, binding sites onsubstrate molecules or receptors, DNA binding sites, or such like. Sinceit is the interactive capacity and nature of a peptide, polypeptide orprotein that defines its biological (e.g., immunological) functionalactivity, certain amino acid sequence substitutions can be made in anamino acid sequence (or, of course, its underlying DNA coding sequence)and nevertheless obtain a peptide or polypeptide with like (agonistic)properties. It is thus contemplated by the inventors that variouschanges may be made in the sequence of an antigenic composition such as,for example a SARS-CoV-2 RBD peptide or polypeptide without appreciableloss of biological utility or activity. In particular cases, one or moreof the potential glycosylation sites of RBD is mutated or deleted and inparticular embodiments there is also one or more other amino acids thatare modified compared to the corresponding wild-type sequence.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of the antigeniccomposition comprises amino molecules that are sequential, without anynon-amino molecule interrupting the sequence of amino molecule residues.In other embodiments, the sequence may comprise one or more non-aminomolecule moieties. In particular embodiments, the sequence of residuesof the antigenic composition may be interrupted by one or more non-aminomolecule moieties.

Accordingly, antigenic compositions may encompass an amino moleculesequence comprising at least one of the 20 common amino acids innaturally synthesized proteins, or at least one modified or unusualamino acid.

In terms of variants that are immunologically functional equivalents, itis well understood by the skilled artisan that, inherent in thedefinition is the concept that there is a limit to the number of changesthat may be made within a defined portion of the molecule and stillresult in a molecule with an acceptable level of equivalentimmunological activity. An immunologically functional equivalent peptideor polypeptide are thus defined herein as those peptide(s) orpolypeptide(s) in which certain, not most or all, of the amino acid(s)may be substituted.

In particular, where a shorter length peptide is concerned, it iscontemplated that fewer amino acid substitutions should be made withinthe given peptide. A longer polypeptide may have an intermediate numberof changes. The full-length protein will have the most tolerance for alarger number of changes. Of course, a plurality of distinctpolypeptides/peptides with different substitutions may easily be madeand used in accordance with the invention.

It also is well understood that where certain residues are shown to beparticularly important to the immunological or structural properties ofa protein or peptide, e.g., residues in binding regions or active sites,such residues may not generally be exchanged. This is an importantconsideration in the present invention, where changes in the antigenicsite should be carefully considered and subsequently tested to ensuremaintenance of immunological function (e.g., antigenicity), wheremaintenance of immunological function is desired. In this manner,functional equivalents are defined herein as those peptides orpolypeptides which maintain a substantial amount of their nativeimmunological activity.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as immunologically functionalequivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein, polypeptide or peptide isgenerally understood in the art (Kyte & Doolittle, 1982, incorporatedherein by reference). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the immunological functional equivalent polypeptideor peptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a immunological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+−0.1); glutamate (+3.0.+−0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

Numerous scientific publications have also been devoted to theprediction of secondary structure, and to the identification of anepitope, from analyses of an amino acid sequence (Chou & Fasman,1974a,b; 1978a,b, 1979). Any of these may be used, if desired, tosupplement the teachings of U.S. Pat. No. 4,554,101.

Moreover, computer programs are currently available to assist withpredicting an antigenic portion and an epitopic core region of one ormore proteins, polypeptides or peptides. Examples include those programsbased upon the Jameson-Wolf analysis (Jameson & Wolf, 1988; Wolf et al.,1988), the program PepPlot (Brutlag et al., 1990; Weinberger et al.,1985), and other new programs for protein tertiary structure prediction(Fetrow & Bryant, 1993). Another commercially available software programcapable of carrying out such analyses is MacVector (IBI, New Haven,Conn.).

In further embodiments, major antigenic determinants of a peptide orpolypeptide may be identified by an empirical approach in which portionsof a nucleic acid encoding a peptide or polypeptide are expressed in arecombinant host, and the resulting peptide(s) or polypeptide(s) testedfor their ability to elicit an immune response. For example, PCR can beused to prepare a range of peptides or polypeptides lacking successivelylonger fragments of the C-terminus of the amino acid sequence. Theimmunoactivity of each of these peptides or polypeptides is determinedto identify those fragments or domains that are immunodominant. Furtherstudies in which only a small number of amino acids are removed at eachiteration then allows the location of the antigenic determinant(s) ofthe peptide or polypeptide to be more precisely determined.

Another method for determining a major antigenic determinant of apeptide or polypeptide is the SPOTs system (Genosys Biotechnologies,Inc., The Woodlands, Tex.). In this method, overlapping peptides aresynthesized on a cellulose membrane, which following synthesis anddeprotection, is screened using a polyclonal or monoclonal antibody. Anantigenic determinant of the peptides or polypeptides which areinitially identified can be further localized by performing subsequentsyntheses of smaller peptides with larger overlaps, and by eventuallyreplacing individual amino acids at each position along theimmunoreactive sequence.

Once one or more such analyses are completed, an antigenic composition,such as for example a peptide or a polypeptide is prepared that containat least the essential features of one or more antigenic determinants.An antigenic composition is then employed in the generation of antiseraagainst the composition, and preferably the antigenic determinant(s).

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA; taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid. Nucleic acids encodingthese antigenic compositions also can be constructed and inserted intoone or more expression vectors by standard methods (Sambrook et al.,1987), for example, using PCR cloning methodology. In addition to thepeptidyl compounds described herein, the inventors also contemplate thatother sterically similar compounds may be formulated to mimic the keyportions of the peptide or polypeptide structure or to interactspecifically with, for example, an antibody. Such compounds, which maybe termed peptidomimetics, may be used in the same manner as a peptideor polypeptide of the invention and hence are also immunologicallyfunctional equivalents.

Certain mimetics that mimic elements of protein secondary structure aredescribed in Johnson et al. (1993). The underlying rationale behind theuse of peptide mimetics is that the peptide backbone of proteins existschiefly to orientate amino acid side chains in such a way as tofacilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is thus designed to permit molecularinteractions similar to the natural molecule.

In particular embodiments, an antigenic composition is mutated forpurposes such as, for example, enhancing its immunogenicity or producingor identifying a immunologically functional equivalent sequence. Methodsof mutagenesis are well known to those of skill in the art (Sambrook etal., 1987).

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In some embodiments, site directed mutagenesis is used. Site-specificmutagenesis is a technique useful in the preparation of an antigeniccomposition, through specific mutagenesis of the underlying DNA. Ingeneral, the technique of site-specific mutagenesis is well known in theart. The technique further provides a ready ability to prepare and testsequence variants, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of amutant through the use of specific oligonucleotide sequence(s) whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the position being mutated. Typically, a primer of about 17 toabout 75 nucleotides in length is preferred, with about 10 to about 25or more residues on both sides of the position being altered, whileprimers of about 17 to about 25 nucleotides in length being morepreferred, with about 5 to 10 residues on both sides of the positionbeing altered.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. As will be appreciated by one of ordinary skill in theart, the technique typically employs a bacteriophage vector that existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

This mutagenic primer is then annealed with the single-stranded DNApreparation, and subjected to DNA polymerizing enzymes such as, forexample, E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected that include recombinant vectors bearing the mutatedsequence arrangement.

Alternatively, a pair of primers may be annealed to two separate strandsof a double stranded vector to simultaneously synthesize bothcorresponding complementary strands with the desired mutation(s) in aPCR reaction. A genetic selection scheme to enrich for clonesincorporating the mutagenic oligonucleotide has been devised (Kunkel etal., 1987). Alternatively, the use of PCR with commercially availablethermostable enzymes such as Taq polymerase may be used to incorporate amutagenic oligonucleotide primer into an amplified DNA fragment that canthen be cloned into an appropriate cloning or expression vector (Tomicet al., 1990; Upender et al., 1995). A PCR™ employing a thermostableligase in addition to a thermostable polymerase also may be used toincorporate a phosphorylated mutagenic oligonucleotide into an amplifiedDNA fragment that may then be cloned into an appropriate cloning orexpression vector (Michael 1994).

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

Additionally, one particularly useful mutagenesis technique is alaninescanning mutagenesis in which a number of residues are substitutedindividually with the amino acid alanine so that the effects of losingside-chain interactions can be determined, while minimizing the risk oflarge-scale perturbations in protein conformation (Cunningham et al.,1989).

In a further embodiment of the invention, one or more vaccine orimmunizing composition components may be entrapped in a lipid complexsuch as, for example, a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991).

In any case, a vaccine component (e.g., an antigenic peptide orpolypeptide) may be isolated and/or purified from the chemical synthesisreagents, cell or cellular components. In a method of producing thevaccine or immunogenic composition component, purification isaccomplished by any appropriate technique that is described herein orwell-known to those of skill in the art (e.g., Sambrook et al., 1987).There is no general requirement that an antigenic composition of thepresent invention or other vaccine component always be provided in theirmost purified state. Indeed, it is contemplated that less substantiallypurified vaccine or immunogenic composition component, which isnonetheless enriched in the desired compound, relative to the naturalstate, will have utility in certain embodiments, such as, for example,total recovery of protein product, or in maintaining the activity of anexpressed protein. However, it is contemplated that inactive productsalso have utility in certain embodiments, such as, e.g., in determiningantigenicity via antibody generation.

The present invention also provides purified, and in certainembodiments, substantially purified vaccines or immunogenic compositioncomponents. The term “purified vaccine component” or “purifiedimmunogenic composition component” as used herein, is intended to referto at least one respective vaccine or immunogenic composition component(e.g., a proteinaceous composition, isolatable from cells), wherein thecomponent is purified to any degree relative to its naturally-obtainablestate, e.g., relative to its purity within a cellular extract orreagents of chemical synthesis. In certain aspects wherein the vaccinecomponent is a proteinaceous composition, a purified vaccine componentalso refers to a wild-type or mutant protein, polypeptide, or peptidefree from the environment in which it naturally occurs.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific compound (e.g., a protein,polypeptide, or peptide) forms the major component of the composition,such as constituting about 50% of the compounds in the composition ormore. In preferred embodiments, a substantially purified vaccinecomponent will constitute more than about 60%, about 70%, about 80%,about 90%, about 95%, about 99% or even more of the compounds in thecomposition.

In certain embodiments, a vaccine or immunogenic composition componentmay be purified to homogeneity. As applied to the present invention,“purified to homogeneity,” means that the vaccine component has a levelof purity where the compound is substantially free from other chemicals,biomolecules or cells. For example, a purified peptide, polypeptide orprotein will often be sufficiently free of other protein components sothat degradative sequencing may be performed successfully. Variousmethods for quantifying the degree of purification of a vaccinecomponent will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificprotein activity of a fraction (e.g., antigenicity), or assessing thenumber of polypeptides within a fraction by gel electrophoresis.

Various techniques suitable for use in chemical, biomolecule orbiological purification, well known to those of skill in the art, may beapplicable to preparation of a vaccine component of the presentinvention. These include, for example, precipitation with ammoniumsulfate, PEG, antibodies and the like or by heat denaturation, followedby centrifugation; fractionation, chromatographic procedures, includingbut not limited to, partition chromatograph (e.g., paper chromatograph,thin-layer chromatograph (TLC), gas-liquid chromatography and gelchromatography) gas chromatography, high performance liquidchromatography, affinity chromatography, supercritical flowchromatography ion exchange, gel filtration, reverse phase,hydroxylapatite, lectin affinity; isoelectric focusing and gelelectrophoresis (see for example, Sambrook et al. 1989; and Freifelder,Physical Biochemistry, Second Edition, pages 238-246, incorporatedherein by reference).

Given many DNA and proteins are known (see for example, the NationalCenter for Biotechnology Information's GenBank and GenPept databases, ormay be identified and amplified using the methods described herein, anypurification method for recombinately expressed nucleic acid orproteinaceous sequences known to those of skill in the art can now beemployed. In certain aspects, a nucleic acid may be purified onpolyacrylamide gels, and/or cesium chloride centrifugation gradients, orby any other means known to one of ordinary skill in the art (see forexample, Sambrook et al. 1989, incorporated herein by reference). Infurther aspects, a purification of a proteinaceous sequence may beconducted by recombinately expressing the sequence as a fusion protein.Such purification methods are routine in the art. This is exemplified bythe generation of an specific protein-glutathione S-transferase fusionprotein, expression in E. coli, and isolation to homogeneity usingaffinity chromatography on glutathione-agarose or the generation of apolyhistidine tag on the N- or C-terminus of the protein, and subsequentpurification using Ni-affinity chromatography. In particular aspects,cells or other components of the vaccine may be purified by flowcytometry. Flow cytometry involves the separation of cells or otherparticles in a liquid sample, and is well known in the art (see, forexample, U.S. Pat. Nos. 3,826,364, 4,284,412, 4,989,977, 4,498,766,5,478,722, 4,857,451, 4,774,189, 4,767,206, 4,714,682, 5,160,974 and4,661,913). Any of these techniques described herein, and combinationsof these and any other techniques known to skilled artisans, may be usedto purify and/or assay the purity of the various chemicals,proteinaceous compounds, nucleic acids, cellular materials and/or cellsthat may comprise a vaccine of the present invention. As is generallyknown in the art, it is believed that the order of conducting thevarious purification steps may be changed, or that certain steps may beomitted, and still result in a suitable method for the preparation of asubstantially purified antigen or other vaccine component.

It is contemplated that an antigenic composition of the invention may becombined with one or more additional components to form a more effectivecomposition or vaccine. Non-limiting examples of additional componentsinclude, for example, one or more additional antigens, immunomodulatorsor adjuvants to stimulate an immune response to an antigenic compositionof the present invention and/or the additional component(s).

For example, in some embodiments one or more immunomodulators can beincluded in the vaccine to augment a cell's or a patient's (e.g., ananimal's) response. Immunomodulators can be included as purifiedproteins, nucleic acids encoding immunomodulators, and/or cells thatexpress immunomodulators in the vaccine composition, for example. Thefollowing sections list non-limiting examples of immunomodulators thatare of interest, and it is contemplated that various combinations ofimmunomodulators may be used in certain embodiments (e.g., a cytokineand a chemokine).

Interleukins, cytokines, nucleic acids encoding interleukins orcytokines, and/or cells expressing such compounds are contemplated aspossible vaccine components. Interleukins and cytokines, include but arenot limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18,.beta.-interferon, α-interferon, γ-interferon, angiostatin,thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, tumornecrosis factor, TGFβ, LT and combinations thereof.

Chemokines, nucleic acids that encode for chemokines, and/or cells thatexpress such also may be used as vaccine components. Chemokinesgenerally act as chemoattractants to recruit immune effector cells tothe site of chemokine expression. It may be advantageous to express aparticular chemokine coding sequence in combination with, for example, acytokine coding sequence, to enhance the recruitment of other immunesystem components to the site of treatment. Such chemokines include, forexample, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinationsthereof. The skilled artisan will recognize that certain cytokines arealso known to have chemoattractant effects and could also be classifiedunder the term chemokines.

In certain embodiments, an antigenic composition may be chemicallycoupled to a carrier or recombinantly expressed with an immunogeniccarrier peptide or polypeptide (e.g., an antigen-carrier fusion peptideor polypeptide) to enhance an immune reaction. Exemplary and preferredimmunogenic carrier amino acid sequences include hepatitis B surfaceantigen, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).Other albumins such as ovalbumin, mouse serum albumin or rabbit serumalbumin also can be used as immunogenic carrier proteins. Means forconjugating a polypeptide or peptide to an immunogenic carrier proteinare well known in the art and include, for example, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

It may be desirable to coadminister biologic response modifiers (BRM),which have been shown to upregulate T cell immunity or downregulatesuppressor cell activity. Such BRMs include, but are not limited to,cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose cyclophosphamide(CYP; 300 mg/m.sup.2) (Johnson/Mead, NJ), or a gene encoding a proteininvolved in one or more immune helper functions, such as B-7.

Immunization protocols have used adjuvants to stimulate responses formany years, and as such adjuvants are well known to one of ordinaryskill in the art. Some adjuvants affect the way in which antigens arepresented. For example, the immune response is increased when proteinantigens are precipitated by alum. Emulsification of antigens alsoprolongs the duration of antigen presentation.

In one aspect, an adjuvant effect is achieved by use of an agent, suchas alum, used in about 0.05 to about 0.1% solution in phosphate bufferedsaline. Alternatively, the antigen is made as an admixture withsynthetic polymers of sugars (Carbopol) used as an about 0.25% solution.Adjuvant effect may also be made my aggregation of the antigen in thevaccine by heat treatment with temperatures ranging between about 70degrees to about 101 degrees C. for a 30-second to 2-minute period,respectively. Aggregation by reactivating with pepsin treated (Fab)antibodies to albumin, mixture with bacterial cell(s) such as C. parvum,an endotoxin or a lipopolysaccharide component of Gram-negativebacteria, emulsion in physiologically acceptable oil vehicles, such asmannide mono-oleate (Aracel A), or emulsion with a 20% solution of aperfluorocarbon (Fluosol-DA®) used as a block substitute, also may beemployed.

Some adjuvants, for example, certain organic molecules obtained frombacteria, act on the host rather than on the antigen. An example ismuramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), abacterial peptidoglycan. The effects of MDP, as with most adjuvants, arenot fully understood. MDP stimulates macrophages but also appears tostimulate B cells directly. The effects of adjuvants, therefore, are notantigen-specific. If they are administered together with a purifiedantigen, however, they can be used to selectively promote the responseto the antigen.

Adjuvants have been used experimentally to promote a generalizedincrease in immunity against unknown antigens (e.g., U.S. Pat. No.4,877,611). In certain embodiments, hemocyanins and hemoerythrins mayalso be used in the invention. The use of hemocyanin from keyhole limpet(KLH) is preferred in certain embodiments, although other molluscan andarthropod hemocyanins and hemoerythrins may be employed.

Various polysaccharide adjuvants may also be used. For example, the useof various pneumococcal polysaccharide adjuvants on the antibodyresponses of mice has been described (Yin et al., 1989). The doses thatproduce optimal responses, or that otherwise do not produce suppression,should be employed as indicated (Yin et al., 1989). Polyamine varietiesof polysaccharides are particularly preferred, such as chitin andchitosan, including deacetylated chitin.

Another group of adjuvants are the muramyl dipeptide (MDP,N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptidederivative of muramyl dipeptide which is described for use in artificialliposomes formed from phosphatidyl choline and phosphatidyl glycerol. Itis the to be effective in activating human monocytes and destroyingtumor cells, but is non-toxic in generally high doses. The compounds ofU.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, arecontemplated for use with cellular carriers and other embodiments of thepresent invention.

Another adjuvant contemplated for use in the present invention is BCG.BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium)and BCG-cell wall skeleton (CWS) may also be used as adjuvants in theinvention, with or without trehalose dimycolate. Trehalose dimycolatemay be used itself. Trehalose dimycolate administration has been shownto correlate with augmented resistance to influenza virus infection inmice (Azuma et al., 1988). Trehalose dimycolate may be prepared asdescribed in U.S. Pat. No. 4,579,945.

BCG is an important clinical tool because of its immunostimulatoryproperties. BCG acts to stimulate the reticulo-endothelial system,activates natural killer cells and increases proliferation ofhematopoietic stem cells. Cell wall extracts of BCG have proven to haveexcellent immune adjuvant activity. Molecular genetic tools and methodsfor mycobacteria have provided the means to introduce foreign genes intoBCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990;Martin et al., 1990).

Live BCG is an effective and safe vaccine used worldwide to preventtuberculosis. BCG and other mycobacteria are highly effective adjuvants,and the immune response to mycobacteria has been studied extensively.With nearly 2 billion immunizations, BCG has a long record of safe usein man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccinesthat can be given at birth, it engenders long-lived immune responseswith only a single dose, and there is a worldwide distribution networkwith experience in BCG vaccination. An exemplary BCG vaccine is sold asTICE BCG (Organon Inc., West Orange, N.J.).

Amphipathic and surface active agents, e.g., saponin and derivativessuch as QS21 (Cambridge Biotech), form yet another group of adjuvantsfor use with the immunogens of the present invention. Nonionic blockcopolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) mayalso be employed. Oligonucleotides are another useful group of adjuvants(Yamamoto et al., 1988). Quil A and lentinen are other adjuvants thatmay be used in certain embodiments of the present invention.

In some embodiments, detoxified endotoxins can be used as adjuvants,such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034.These refined detoxified endotoxins are effective in producing adjuvantresponses in mammals. Of course, the detoxified endotoxins may becombined with other adjuvants to prepare multi-adjuvant-incorporatedcells. For example, combination of detoxified endotoxins with trehalosedimycolate is particularly contemplated, as described in U.S. Pat. No.4,435,386. Combinations of detoxified endotoxins with trehalosedimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No.4,505,899), as is combination of detoxified endotoxins with cell wallskeleton (CWS) or CWS and trehalose dimycolate, as described in U.S.Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWSand trehalose dimycolate, without detoxified endotoxins, is alsoenvisioned to be useful, as described in U.S. Pat. No. 4,520,019.

In other embodiments, the present invention contemplates that a varietyof adjuvants may be employed in the membranes of cells, resulting in animproved immunogenic composition. The only requirement is, generally,that the adjuvant be capable of incorporation into, physical associationwith, or conjugation to, the cell membrane of the cell in question.Those of skill in the art will know the different kinds of adjuvantsthat can be conjugated to cellular vaccines in accordance with thisinvention and these include alkyl lysophosphilipids (ALP); BCG; andbiotin (including biotinylated derivatives) among others. Certainadjuvants particularly contemplated for use are the teichoic acids fromGram-cells. These include the lipoteichoic acids (LTA), ribitol teichoicacids (RTA) and glycerol teichoic acid (GTA). Active forms of theirsynthetic counterparts may also be employed in connection with theinvention (Takada et al., 1995a).

Various adjuvants, even those that are not commonly used in humans, maystill be employed in animals, where, for example, one desires to raiseantibodies or to subsequently obtain activated T cells. The toxicity orother adverse effects that may result from either the adjuvant or thecells, e.g., as may occur using non-irradiated tumor cells, isirrelevant in such circumstances.

One group of adjuvants preferred for use in some embodiments of thepresent invention are those that can be encoded by a nucleic acid (e.g.,DNA or RNA). It is contemplated that such adjuvants may be encoded in anucleic acid (e.g., an expression vector) encoding the antigen, or in aseparate vector or other construct. These nucleic acids encoding theadjuvants can be delivered directly, such as for example with lipids orliposomes.

An antigenic composition of the present invention may be mixed with oneor more additional components (e.g., excipients, salts, etc.) which arepharmaceutically acceptable and compatible with at least one activeingredient (e.g., antigen). Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol and combinations thereof.

An antigenic composition of the present invention may be formulated intothe vaccine as a neutral or salt form. A pharmaceutically-acceptablesalt, includes the acid addition salts (formed with the free aminogroups of the peptide) and those which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acid, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. A salt formedwith a free carboxyl group also may be derived from an inorganic basesuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxide, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, procaine, and combinations thereof.

In addition, if desired, an antigenic composition may comprise minoramounts of one or more auxiliary substances such as for example wettingor emulsifying agents, pH buffering agents, etc. which enhance theeffectiveness of the antigenic composition or vaccine.

Once produced, synthesized and/or purified, an antigen or other vaccinecomponent may be prepared as a vaccine or immunogenic composition foradministration to an individual. The preparation of a vaccine isgenerally well understood in the art, as exemplified by U.S. Pat. Nos.4,608,251, 4,601,903, 4,599,231, 4,599,230, and 4,596,792, allincorporated herein by reference. Such methods may be used to prepare avaccine comprising an antigenic composition comprising a particular RBDof SARS-CoV-2 as active ingredient(s), in light of the presentdisclosure. In particular embodiments, the compositions of the presentinvention are prepared to be pharmacologically acceptable vaccines.

In some embodiments, pharmaceutical vaccine or immunogenic compositionsof the present invention comprise an effective amount of one or morecertain RBDs of SARS-CoV-2 dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains at least one RBD of SARS-CoV-2 will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). In some embodiments, the antigen, such as the RBD ofSARS-CoV-2 may comprise different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.Except insofar as any conventional carrier is incompatible with theactive ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

In some embodiments, the antigen, such as the RBD of SARS-CoV-2 may beformulated into a composition in a free base, neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts,e.g., those formed with the free amino groups of a proteinaceouscomposition, or which are formed with inorganic acids such as forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In some embodiments, sterile injectable solutions can be prepared byincorporating the antigens in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and/or theother ingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less than 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

For a broad overview of controlled delivery systems, see, Banga, A. J.,Therapeutic Peptides and Proteins: Formulation, Processing, and DeliverySystems, Technomic Publishing Company, Inc., Lancaster, Pa., (1995).Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nano spheres, and nanoparticles. Microcapsules can containthe therapeutically active agents as a central core. In microspheres thetherapeutic can be dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Microparticles are typically around 100 μm in diameter.See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J.Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994);and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus,ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, (1992).

In some embodiments, polymers can be used for controlled release ofcompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). In yet another aspect, liposomes can be used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)).

A vaccination or immunizing composition delivery schedule and dosagesmay be varied on a patient by patient basis, taking into account, forexample, factors such as the weight and age of the patient, the type ofdisease being treated, the severity of the disease condition, previousor concurrent therapeutic interventions, the manner of administrationand the like, which can be readily determined by one of ordinary skillin the art.

A vaccine or immunizing composition may be administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and immunogenic. For example, theintramuscular route may be preferred in the case of toxins with shorthalf lives in vivo. The quantity to be administered depends on thesubject to be treated, including, e.g., the capacity of the individual'simmune system to synthesize antibodies, and the degree of protectiondesired. The dosage of the vaccine will depend on the route ofadministration and will vary according to the size of the host. Preciseamounts of an active ingredient required to be administered depend onthe judgment of the practitioner. In certain embodiments, pharmaceuticalcompositions may comprise, for example, at least about 0.1% of an activecompound. In other embodiments, the active compound may comprise betweenabout 2% to about 75% of the weight of the unit, or between about 25% toabout 60%, for example, and any range derivable therein. However, asuitable dosage range may be, for example, of the order of severalhundred micrograms active ingredient per vaccination. Proper dosages ofthe polypeptides or heat killed or attenuated pathogens can bedetermined without undue experimentation using standard dose-responseprotocols. In other non-limiting examples, a dose may also comprise fromabout 1 microgram/kg/body weight, about 5 microgram/kg/body weight,about 10 microgram/kg/body weight, about 50 microgram/kg/body weight,about 100 microgram/kg/body weight, about 200 microgram/kg/body weight,about 350 microgram/kg/body weight, about 500 microgram/kg/body weight,about 1 milligram/kg/body weight, about 5 milligram/kg/body weight,about 10 milligram/kg/body weight, about 50 milligram/kg/body weight,about 100 milligram/kg/body weight, about 200 milligram/kg/body weight,about 350 milligram/kg/body weight, about 500 milligram/kg/body weight,to about 1000 mg/kg/body weight or more per vaccination, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above. A suitable regime for initial administrationand booster administrations (e.g., innoculations) are also variable, butare typified by an initial administration followed by subsequentinoculation(s) or other administration(s).

In many instances, it will be desirable to have multiple administrationsof the vaccine or immunizing composition, usually not exceeding sixvaccinations, for example, more usually not exceeding four vaccinationsand in some cases one or more, usually at least about threevaccinations. The vaccinations may be at from two to twelve-weekintervals, more usually from three to five week intervals, althoughlonger intervals are encompassed herein. Periodic boosters at intervalsof 1-5 years, usually three years, may be desirable to maintainprotective levels of the antibodies.

The course of the immunization may be followed by assays for antibodiesfor the supernatant antigens. The assays may be performed by labelingwith conventional labels, such as radionuclides, enzymes, fluorescents,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays. Other immune assayscan be performed and assays of protection from challenge with the RBD ofSARS-CoV-2 can be performed, following immunization.

Any of the compositions and devices described herein may be comprised ina kit. In a non-limiting example, an RBD SARS-CoV-2 spike compositionmay be comprised in a kit along with the microneedle delivery device.The immunizing components of the kit may be packaged either in aqueousmedia or in lyophilized form. The kits of the present invention alsowill typically include a means for containing the composition and anyother reagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

The component(s) of the kit may be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means. Thekits may comprise a container means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

As provided herein, the immunizing composition is administered using amicroneedle delivery device.

In some embodiments, the microneedle delivery device is shown in FIG.7-12 or 14-22. In some embodiments, the device comprises a cap, ahousing, and a cartridge. See FIG. 9.

Cap

The cap (see FIG. 10(1)) covers the housing and microneedle head, e.g.,to prevent entry of dust, microbes and other foreign particles. In someembodiments, the cap and housing come together and are sealed. The capcan be removed just before the administration of the therapeuticcomposition. In some embodiments, the cap is made from a substancecomprising polycarbonate.

Housing

In some embodiments, the housing comprises of the Cover (FIG. 16 (2)),Microneedle array (FIG. 11 (3)), Carrier body (FIG. 11 (4)), Punctureneedle (FIG. 11 (5)), Pogo pin (FIG. 11 (6)), O-Ring (FIG. 11 (7)).

In some embodiments the housing comprises a microneedle array comprisinga single microchannel or microneedle or it can comprise a plurality ofmicrochannels or microneedles. In some embodiments, they are hollow ornon-hollow, wherein one or multiple grooves are inset along an outerwall of the microneedles/microchannels. In some embodiments, thecomposition can be delivered into the skin by passing through the one ormultiple grooves along the outer wall of the microneedle/microchannel.In some embodiments, a single groove inset along the outer wall of themicroneedle has a screw thread shape going clockwise or counterclockwisearound the microneedle/microchannel.

In some embodiments, the microneedles are from 0.1 mm to about 2.5 mm inlength and from 0.01 mm to about 0.05 mm in diameter. In someembodiments, the microneedles are made from a substance comprising gold.

In some embodiments, they can also be made of 24-carat gold platedstainless steel.

In some embodiments, the plurality of microneedles comprises an array ofmicroneedles in the shape of a circle or a four-sided figure.

In some embodiments, the puncture needle is made from a substancecomprising stainless steel.

In some embodiments, when the apparatus is operated, the puncture needlepunctures the cartridge cap enabling the release of composition into thehousing

In some embodiments, the O-Ring and puncture needle gets pushed towardthe microneedle array after puncturing the cartridge cap thus disablingthe further flow of composition and repeated puncturing process.

In some embodiments, the O-Ring breaks automatically on forced reuse. Insome embodiments, the housing encourages flow of the compositioncontained in the cartridge into the skin.

Cartridge

In some embodiments, the cartridge comprises a lower body (FIG. 12 (8)),lower plate (FIG. 12 (9)), cartridge main body (FIG. 12 (10)), andcartridge cap (FIG. 12 (11)).

In some embodiments, the cartridge main body is transparent made up ofglass or other sterilizable container materials and contains thecomposition. The cartridge holds the composition to be delivered.

In some embodiments, the composition can be an FDA approved drug or ainactivated/attenuated pathogen, an immunologically-effective amount ofone or more polypeptides from a pathogen, antigenic fragments, orvariants thereof.

In some embodiments, the pathogen is Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2), which causes the disease COVID-19.

In some embodiments, the polypeptide comprises SEQ ID NO:4 or aminoacids 330 to 521 of SEQ ID NO:4. In some embodiments, the antigencomprises a fragment of SEQ ID NO:4.

In some embodiments, the composition comprises one or more adjuvants.

In some embodiments, the composition must be stable under the conditionsof manufacture and storage, and preserved against the contaminatingaction of microorganisms, such as bacteria and fungi.

In some embodiments, the composition can also be prolonged absorption ofan injectable composition which can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In some embodiments, the composition can also include microspheres,microparticles, microcapsules, nanocapsules, nanospheres, andnanoparticles. Microcapsules can contain the therapeutically activeagents as a central core. In microspheres the therapeutic can bedispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 micron are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively.Microparticles are typically around 100 microns in diameter.

In some embodiments, the composition can also include polymers that canenable controlled release of compositions disclosed herein. It can bedegradable or nondegradable polymeric matrices.

In some embodiments, the when the apparatus is operated, the cartridgecap is punctured by the puncture needle in the housing enabling therelease of composition into the housing.

In some embodiments, the invention provides a system for massdistribution of pharmaceutical compositions using self-administeredsmart apparatuses in a pandemic. In some embodiments, the system cancomprise one or more of the following steps. See also FIG. 13.

-   -   1. In some embodiments, the apparatus is delivered to houses,        cities and pick-up locations through mass-distribution        mechanisms or they are dispatched through aerial vehicles such        as helicopters, drones, and other unmanned aerial vehicles.    -   2. In some embodiments, the user collects the apparatus.    -   3. In some embodiments, the user removes the packaging over the        apparatus.    -   4. In some embodiments, the apparatus may contain pre-loaded        composition or the composition can be loaded by the user.    -   5. In some embodiments, the if composition is not preloaded, the        composition is loaded into the cartridge by a vacuum sack that        may be provided with the material.    -   6. In some embodiments, the user visually inspects the        transparent cartridge to ensure the composition is present.    -   7. In some embodiments, the user administers the apparatus by a        repeated motion of penetrating the microneedle delivery        apparatus into the subject's skin. In some embodiments, it can        be in different areas of the subject's body such as the        subject's skin in the head, limbs and/or torso regions. In some        embodiments, it can also be in areas with close proximity to one        or more lymph nodes.    -   8. In some embodiments, after administration, the mechanical        spring-loaded system gets locked thus disabling the flow of        composition into the microneedles.    -   9. In some embodiments, if the user attempts forced reuse, the        housing breaks automatically.    -   10. In some embodiments, the user can dispose of the apparatus        after it's used. The user can also replace the housing and/or        cartridge.    -   11. In some embodiments, the apparatus is marked with a modular        smart label data transmission system that helps with tracking of        individual apparatus and helps collect data and feedback to        optimize user experience and operations.

In some embodiments, the microneedle delivery device useful in themethods of the invention is depicted in FIG. 7. In some embodiments, themicroneedle head in the microneedle drug delivery device is such asdescribed in Korean Patent No. 10-1582822, which is incorporated byreference herein in its entirety.

In some embodiments, the microneedle delivery device comprises

-   -   i) a plurality of microneedles, wherein the microneedles are        hollow or non-hollow, wherein one or multiple grooves are inset        along an outer wall of the microneedles; and    -   ii) a reservoir that holds the composition to be delivered,        wherein the reservoir is attached to or contains a means to        encourage flow of the bioactive composition contained in the        reservoir into the skin.

In some embodiments, the means to encourage flow of the compositioncontained in the reservoir into the skin is selected from the groupconsisting of a plunger, pump, and suction mechanism. In someembodiments, the means to encourage flow of the composition contained inthe reservoir into the skin is a mechanical spring-loaded pump system.

In some embodiments, the microneedles have a single groove inset alongthe outer wall of the microneedle, wherein the single groove has a screwthread shape going clockwise or counterclockwise around the microneedle.

In some embodiments, the microneedles are from 0.1 mm to about 2.5 mm inlength and from 0.01 mm to about 0.05 mm in diameter.

In some embodiments, the microneedles are made from a substancecomprising gold.

In some embodiments, the plurality of microneedles comprises an array ofmicroneedles in the shape of a circle.

In some embodiments, the microneedles are made of 24-carat gold platedstainless steel and comprise an array of about 10 to about 50microneedles. In some embodiments, the array comprises 20 microneedles.

In some embodiments, the microneedle delivery device is repeatedlypressed against the subject's skin to deliver the composition to thearea of the skin to be treated. In some embodiments, the microneedledelivery device is repeatedly pressed about 10, about 20, about 30,about 40, about 50, about 100, about 200, about 300, about 400, about500, about 600, about 700, about 800, about 900, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1600, about 1700,about 1800, about 1900, or about 2000 or more times to administer thecomposition.

In some embodiments, the immunizing composition is administered by themicroneedle delivery device with a repeated motion of penetrating themicroneedle delivery device into the skin of the subject. In someembodiments, the composition is delivered into the skin by passingthrough the one or multiple grooves along the outer wall of themicroneedle. In some embodiments, the microneedles are non-hollow.

In some embodiments, the administering comprises a repeated motion ofpenetrating the microneedle delivery device into the subject's skin indifferent areas of the subject's body.

In some embodiments, the subject's skin in the head, limbs and/or torsoregions are repeatedly penetrated by the microneedle delivery device. Insome embodiments, the subject's skin is penetrated in regions that arein proximity to one or more lymph nodes.

For example, repeated penetrations can be made in the subject's arms,legs, and torso in order to deliver the immunizing composition todifferent areas of the subject's body, in order to enhance the subject'simmune response.

In some embodiments, the microneedle delivery device comprises a singleor an array of microneedles. In some embodiments, the microneedles willhave one or multiple grooves inset along its outer wall. This structuralfeature of the dermal delivery device allows liquids stored in areservoir at the base of each needle to travel along the needle shaftinto the tissue.

In some embodiments, the microneedle array comprises from about 1 toabout 500 microneedles, which will be anywhere from about 0.1 to about2.5 mm in length and from 0.01 to about 0.5 mm in diameter, and becomposed of any metal, metal alloy, metalloid, polymer, or combinationthereof, such as gold, steel, silicon, PVP (polyvinylpyrrlidone), etc.The microneedles will each have one or more recesses running a certaindepth into the outer wall to allow for flow of the substance to bedelivered down the microneedle and into the dermis; these recesses canbe in a plurality of shapes, including but not limited to: straightline, cross shape (+), flat shape (−), or screw thread shape goingclockwise or counterclockwise. The array will be in any shape orcombination of shapes, continuous, or discontinuous. The list ofpossible shapes includes, but is not limited to, circles, triangles,rectangles, squares, rhomboids, trapezoids, and any other regular orirregular polygons. The array can be attached to a reservoir to hold thesubstances to be delivered, and this reservoir will be any volume (0.25mL to 5 mL), shape, color, or material (glass, metal, alloy, orpolymer), as determined necessary. This reservoir will itself beattached to or contain a means to encourage flow of the drug solutionscontained in the reservoir into the skin. Two non-limiting examples ofsuch means are 1) a plate and spring that allows the contained solutionsto flow only when the device is tapped into the skin, and 2) a syringethat contains the drug solutions to be delivered and includes a plungerthat can be depressed to mechanically drive the solution into the skin.

The microneedle delivery device is capable of delivering compositionsdirectly to the epidermal, dermal and subcuticular layers of the skin.Therefore, it should be understood that further embodiments developedfor use with non-hollow or hollow microneedle systems of delivery bythose skilled in the art fall within the spirit and scope of thisdisclosure.

In another aspect, a microneedle device for use in the methods describedherein is a device such as described in U.S. Pat. No. 8,257,324, whichis hereby incorporated by reference. Briefly, the devices include asubstrate to which a plurality of hollow microneedles are attached orintegrated, and at least one reservoir, containing a bioactiveformulation, selectably in communication with the microneedles, whereinthe volume or amount of composition to be delivered can be selectivelyaltered. The reservoir can be, for example, formed of a deformable,preferably elastic, material. The device typically includes a means,such as a plunger, for compressing the reservoir to drive the bioactiveformulation from the reservoir through the microneedles, A reservoir,can be, for example, a syringe or pump connected to the substrate. Adevice, in some instances, comprises: a plurality of hollow microneedles(each having a base end and a tip), with at least one hollow pathwaydisposed at or between the base end and the tip, wherein themicroneedles comprise a metal; a substrate to which the base ends of themicroneedles are attached or integrated; at least one reservoir in whichthe material is disposed and which is in connection with the base end ofat least one of the microneedles, either integrally or separably; asealing mechanism interposed between the at least one reservoir and thesubstrate, wherein the sealing mechanism comprises a fracturablebarrier; and a device that expels the material in the reservoir into thebase end of at least one of the microneedles and into the skin. Thereservoir comprises a syringe secured to the substrate, and the devicethat expels the material comprises a plunger connected to a top surfaceof the reservoir. The substrate may be adapted to removably connect to astandard or Luer-lock syringe. In one instance, the device may furtherinclude a spring engaged with the plunger. In another instance, thedevice may further include an attachment mechanism that secures thesyringe to the device. In another instance, the device may furtherinclude a sealing mechanism that is secured to the tips of themicroneedles. In another instance, the device may further include meansfor providing feedback to indicate that delivery of the material fromthe reservoir has been initiated or completed. An osmotic pump may beincluded to expel the material from the reservoir. A plurality ofmicroneedles may be disposed at an angle other than perpendicular to thesubstrate. In certain instances, the at least one reservoir comprisesmultiple reservoirs that can be connected to or are in communicationwith each other. The multiple reservoirs may comprise a first reservoirand a second reservoir, wherein the first reservoir contains a solidformulation and the second reservoir contains a liquid carrier for thesolid formulation. A fracturable barrier for use in the devices can be,for example, a thin foil, a polymer, a laminate film, or a biodegradablepolymer. The device may further comprise, in some instances, means forproviding feedback to indicate that the microneedles have penetrated theskin.

In some embodiments, the device can include, in some instances, a singleor plurality of solid, screw-type microneedles, of single or variedlength. Typically the needles attach to a substrate or are embeddedwithin the substrate. The substrate can be made of any metal, metalalloy, ceramics, organics metalloid, polymer, or combination thereof,including composites, such as gold, steel, silicon, PVP(polyvinylpyrrlidone) etc. The screw-shape dimensions may be variable.For example, in one embodiment the screw-shape may be a tight coiledscrew shape, whereas in another embodiment the screw-shape might be aloose coiled screw shape whereby the screw threads in one embodiment lieclosely together along the outer edge of the needle and, in anotherembodiment, the screw threads lie far from each other along the outeredge of the needle.

In one embodiment a reservoir would attach to the substrate to allowdrug solution to flow down the side of the microneedles. In oneembodiment the reservoir is a solid canister of differing sizesdepending on the desired volume or amount of drug to be delivered. Thereservoir contains the drug to be delivered. In another embodiment, thereservoir can be supported by a mechanical (spring loaded or electrifiedmachine-driven) pump system to deliver the drug solution. In anotherembodiment, the reservoir is composed of a rubber, elastic, or otherwisedeformable and flexible material to allow manual squeezing to deliverthe drug solution. In another embodiment the device includes hollowneedles or needles with alternative ridges and shapes to moreefficiently drive solution from the reservoir through to the dermis.

A device described herein may contain, in certain instances, abouttwenty screw thread design surgical grade microneedles. Each microneedlehas a diameter that is thinner than a human hair and may be used fordirect dermal application. In one instance, a microneedle has a diameterof less than about 0.18 mm. In another instance, a microneedle has adiameter of about 0.15 mm, about 0.14 mm, about 0.13 mm, about 0.12 mm,about 0.11 mm, or about 0.10 mm. Each microneedle may be plated with 24carat gold. The device allows for targeted and uniform delivery of acomposition comprising the immunizing composition into the skin in aprocess that is painless compared to injectables. Administration canresult in easy and precise delivery of a composition comprising theimmunizing composition with generally no bruising, pain, swelling, andbleeding caused by the injection.

The device may include means, manual or mechanical, for compressing thereservoir, creating a vacuum, or otherwise using gravity or pressure todrive the immunizing composition from the reservoir through themicroneedles or down along the sides of the microneedle. The means caninclude a plunger, pump or suction mechanism. In another embodiment, thereservoir further includes a means for controlling rate and precisequantity of drug delivered by utilizing a semi-permeable membrane, toregulate the rate or extent of drug which flows along the shaft of themicroneedles. The microneedle device enhances transportation of drugsacross or into the tissue at a useful rate. For example, the microneedledevice must be capable of delivering drug at a rate sufficient to betherapeutically useful. The rate of delivery of the drug composition canbe controlled by altering one or more of several design variables. Forexample, the amount of material flowing through the needles can becontrolled by manipulating the effective hydrodynamic conductivity (thevolumetric through-capacity) of a single device array, for example, byusing more or fewer microneedles, by increasing or decreasing the numberor diameter of the bores in the microneedles, or by filling at leastsome of the microneedle bores with a diffusion-limiting material. It canbe preferred, however, to simplify the manufacturing process by limitingthe needle design to two or three “sizes” of microneedle arrays toaccommodate, for example small, medium, and large volumetric flows, forwhich the delivery rate is controlled by other means.

Other means for controlling the rate of delivery include varying thedriving force applied to the drug composition in the reservoir. Forexample, in passive diffusion systems, the concentration of drug in thereservoir can be increased to increase the rate of mass transfer. Inactive systems, for example, the pressure applied to the reservoir canbe varied, such as by varying the spring constant or number of springsor elastic bands. In either active or passive systems, the barriermaterial can be selected to provide a particular rate of diffusion forthe drug molecules being delivered through the barrier at the needleinlet.

The array may be in any shape or combination of shapes, continuous, ordiscontinuous. The list of possible shapes includes, but is not limitedto, circles, triangles, rectangles, squares, rhomboids, trapezoids, andany other regular or irregular polygons.

The array may be attached to a reservoir to hold the substances to bedelivered, and this reservoir may be any volume (about 0.25 mL to about5 mL), shape, color, or material (glass, metal, alloy, or polymer), asdetermined necessary.

This reservoir can itself be attached to or contain a means to encourageflow of the drug solutions contained in the reservoir into the skin. Twonon-limiting examples of such means are 1) a plate and spring thatallows the contained solutions to flow only when the device is tappedinto the skin, and 2) a syringe that contains the drug solutions to bedelivered and includes a plunger that can be depressed to mechanicallydrive the solution into the skin.

In some embodiments, the device can include a single or plurality ofsolid, screw-type microneedles, of single or varied lengths housed in aplastic or polymer composite head which embodies a corrugated rubberconnector. In some embodiments, the needles attach to a substrate or areembedded within the substrate. The substrate can be made of any metal,metal alloy, ceramics, organics metalloid, polymer, or combinationthereof, including composites, such as gold, steel, silicon, PVP(polyvinylpyrrlidone) etc. The screw-shape dimensions may be variable.For example, in one embodiment the screw-shape may be a tight coiledscrew shape, whereas in another embodiment the screw-shape might be aloose coiled screw shape. The corrugated rubber connector is a uniqueadvantage conferring feature which bestows the microneedle head with auniversally adoptable feature for interfacing the micro needlecartridges with multiple glass and or plastic vials, reservoirs andcontainers as well as electronic appendages for an altogether enhancedadjunct liquid handling, security and surveillance utility.

In one embodiment a reservoir would attach to the substrate to allowdrug solution to flow down the side of the microneedles. In oneembodiment the reservoir is a solid canister of differing sizesdepending on the desired volume or amount of drug to be delivered. Thereservoir contains the drug to be delivered. In another embodiment, thereservoir can be supported by a mechanical (spring loaded or electrifiedmachine-driven) pump system to deliver the drug solution. In anotherembodiment, the reservoir is composed of a rubber, elastic, or otherwisedeformable and flexible material to allow manual squeezing to deliverthe drug solution. In another embodiment the device includes hollowneedles or needles with alternative ridges and shapes to moreefficiently drive solution from the reservoir through to the dermis.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

We claim:
 1. A method for generating an immune response in a subject,comprising administering to the subject's skin an immunizing compositioncomprising an immunologically-effective amount of i) a polypeptide; orii) a nucleic acid encoding the polypeptide, wherein the polypeptideconsists essentially of a fragment of SEQ ID NO:4, wherein the fragmentconsists of amino acids 330 to 521 of SEQ ID NO:4, wherein theimmunizing composition further comprises an effective amount of anadjuvant when the composition comprises the polypeptide of i), whereinthe composition further comprises lipids when the composition comprisesthe nucleic acid of ii), wherein the composition is administered with amicroneedle delivery device, wherein the microneedle delivery devicecomprises a plurality of microneedles, wherein the microneedles arenon-hollow, wherein the microneedles have a single groove inset alongthe outer wall of the microneedle, wherein the single groove has a screwthread shape going clockwise or counterclockwise around the microneedle;and a reservoir that holds the composition to be delivered, wherein thereservoir is attached to or contains a means to encourage flow of thecomposition contained in the reservoir into the skin; wherein thecomposition is delivered into the skin by passing through the one ormultiple grooves along the outer wall of the microneedle, wherein theadministering comprises a repeated motion of penetrating the microneedledelivery device into the subject's skin.
 2. The method of claim 1,wherein the administering comprises a repeated motion of penetrating themicroneedle delivery device into the subject's skin in different areasof the subject's body.
 3. The method of claim 2, wherein the subject'sskin in the head, limbs and/or torso regions are repeatedly penetratedby the microneedle delivery device.
 4. The method of claim 1, whereinthe subject's skin is penetrated in regions that are in proximity to oneor more lymph nodes.
 5. The method of claim 1, wherein the means toencourage flow of the composition contained in the reservoir into theskin is selected from the group consisting of a plunger, pump andsuction mechanism.
 6. The method of claim 1, wherein the means toencourage flow of the composition contained in the reservoir into theskin is a mechanical spring loaded pump system.
 7. The method of claim1, wherein the microneedles are from 0.1 mm to about 2.5 mm in lengthand from 0.01 mm to about 0.05 mm in diameter.
 8. The method of claim 1,wherein the microneedles are made from a substance comprising gold. 9.The method of claim 1, wherein the plurality of microneedles comprisesan array of microneedles in the shape of a circle.
 10. The method ofclaim 1, wherein the microneedles are made of 24-carat gold platedstainless steel and comprise an array of 20 microneedles.
 11. The methodof claim 1, wherein the subject administers the immunizing compositionto his or her own skin.
 12. A microneedle delivery device comprising animmunizing composition of claim 1.